In normal mammary development, the hormone prolactin (PRL) is critical for alveolar proliferation and differentiation. Increasing evidence supports the involvement of PRL in breast cancer, the leading type of cancer in women and the second leading cause (after lung cancer) of cancer death among women. In 2002, 203,500 new cases of breast cancer were expected to be diagnosed, and 39,600 women were expected to die of the disease. The prolactin receptor (PRLR) is detected in 80% of human breast cancers, and is overexpressed in breast cancer cells. Normal and tumor mammary epithelial cells synthesize PRL and PRLR, thus the PRL could behave as an autocrine growth factor for human breast cancer cells. These results suggest the need for a more complete understanding of PRLR signaling to growth promoting, anti-apoptotic pathways. Tyrosine (Tyr) kinase JAK2 was identified as a PRLR-bound signaling molecule. Identification of the proteins recruited to the PRLR-JAK2 and dissection of the signaling pathways that are subsequently activated will ultimately provide a basis for understanding PRL action. Preliminary data demonstrate that the serine-threonine kinase PAK1 associates with and is Tyr phosphorylated by JAK2. Two-dimensional peptide mapping identified three Tyr(s) of PAK1 which are phosphorylated by JAK2. Tyr phosphorylation of PAK1 by JAK2 was also shown to protect cells from apoptosis. This grant proposes to examine the hypothesis that PAK1 is a substrate for JAK2 and that in response to PRL, PAK1 is activated by JAK2-dependent Tyr phosphorylation and enhances PRL-dependent cell survival. Aim1 will verify that PRL promotes Tyr phosphorylation of PAK1 in vivo. Aim2 will determine whether JAK2 phosphorylation of PAK1 alters PAK1 kinase activity and/or ability of PAK1 to bind PAK1 targets. In Aim3 the effect of JAK2 Tyr phosphorylation of PAK1 on PRL-dependent cell survival will be determined. Because both PAK1 and PRL have been implicated in breast cancer, the proposed studies may ultimately fill out the existing gap between upstream PRL-PRLR-JAK2 events and downstream PAK1-dependent functions in our understanding of the mechanism of human breast cancer. Tyr phosphorylation of PAK1 by JAK2 is likely to represent a novel molecular target in the search for the etiology and treatment of human breast cancer. [unreadable] [unreadable] [unreadable]